This Project will define the mechanisms by which human embryonic stem cell (hESC) self-renewal is preserved and how early differentiation decisions are made. These studies focus on the role of PI3K in promoting GSK3-beta activity, a point of regulation that is critical for blocking an epithelial to mesenchymal transition (EMT) and differentiation towards mesendoderm. Control of mesendoderm development, following loss of PI3K and GSK3-beta activity, is not well understood. To address this issue, respective roles for Wnt and TGF-beta family members in mesendoderm specification will be defined. Understanding these issues is critical not only in relation to hESC self-renewal, but also for understanding the basic mechanisms underpinning early cell fate decisions, including definitive endoderm and mesoderm specification from a mesendoderm precursor. The first Aim of this Project will investigate the role of GSK3-beta in control of EMTs and how it regulates the activity of two transcription factors, Snail 1 and beta-catenin. Mechanisms by which these transcription factors control EMTs will be defined. The second Aim will investigate the signaling pathways required for hESC self-renewal and specifically, how PI3K maintains GSK3-beta activity and inhibits an EMT. The third Aim, will establish the exact conditions for early cell fate commitment to mesendoderm and how Wnt and TGF-beta synergize to pattern this cell type. Finally, we will investigate the use of GSK3-beta inhibitors as compounds that can promote uniform differentiation of hESCs. Understanding the mechanisms of hESC self-renewal is critical if we are to harness their full potential as a developmental model and as a therapeutic source of cells that can be used in regenerative medicine. Our understanding of hESC differentiation into different lineages is only poorly understood. This proposal will focus on a very early stage of cell fate commitment that is critical for differentiation into two key lineages. First, mesoderm which can give rise to blood, muscle and bone. Second, definitive endoderm which gives rise to pancreas, liver, lung, intestine and thyroid.